1. Field of the Invention.
This invention relates to immunoassay of an analyte and materials used therein, and more particularly relates to a method and materials for homogeneous immunoassay using the enzymatic activity of the first component of complement.
2. Description of the Prior Art.
A variety of assay systems which are both rapid and sensitive have been developed to determine the concentration of a substance in a fluid. Immunoassays depend on the binding of an antigen or hapten to a specific antibody and have been particularly useful because they give high levels of specificity and sensitivity. These assays generally employ one of the above reagents in labeled form, the labeled reagent often being referred to as the tracer. Immunoassay procedures may be carried out in solution or on a solid support and may be either heterogeneous or homogeneous. Heterogeneous assays require a separation of bound tracer from free (unbound) tracer. Homogeneous assays do not require a separation step and thereby provide significant advantages in speed, convenience and ease of automation over heterogeneous assays.
Radioimmunoassay (RIA) procedures use radioisotopes as labels, provide high levels of sensitivity and reproducibility, and are amenable to automation for rapid processing of large numbers of samples. However, all RIA procedures require a separation step, since the parameter measured (nuclear decay) cannot be controlled by changing assay conditions or components. In addition, isotopes are costly, have relatively short shelf lives, require expensive and complex equipment, and extensive safety measures for their handling and disposal must be followed.
Fluoroimmunoassay (FIA) uses fluorochromes as labels, provides direct detection of the label, and is readily adaptable to homogeneous assay procedures. However, known homogeneous FIA methods using organic fluorochromes, such as fluorescein or rhodamine derivatives, have not achieved the high sensitivity of RIA, largely because of light scattering by impurities suspended in the assay medium and by background fluorescence emission from other fluorescent materials present in the assay medium.
Enzymes have also been used as labels in immunoassay. In conventional enzyme immunoassay (EIA), an enzyme is covalently conjugated with one component of a specifically binding antigen-antibody pair, and the resulting enzyme conjugate is reacted with a substrate to produce a color which is measured. In U.S. Pat. No. 3,654,090 to Schuurs et al., the unconjugated component is immobilized on a solid support.
In U.S. Pat. No. 4,235,960, Sasse et al. disclose a competitive EIA in which a bound analyte-antibody complex is linked through a bridging second antibody to a third antibody carrying an immunologically attached enzyme label.
U.S. Pat. No. 4,463,090 to Harris teaches increased sensitivity in EIA by cascade amplification. In this procedure, a conjugated enzyme catalytically activates a ligand which either reacts with a substrate to produce a color or activates a second ligand to react. Two or more activation stages may be used.
U.S. Pat. No. 4,342,826 to Cole teaches an improved EIA wherein antigen-tagged liposomes containing encapsulated enzyme label are specifically bound to antibodies, and the liposomes are ruptured by active complement to release the enzyme label.
U.S. Pat. No. 4,138,213 to Masson et al. discloses detection or determination of antigens or antibodies wherein isolated complement subcomponent C1q agglomerates antibody-antigen complexes. Excess C1q in supernatent fractions is measured by addition of a second antibody-antigen complex which carries a radioactive, fluorescent or enzyme label.